EP3714014B1 - Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt - Google Patents
Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt Download PDFInfo
- Publication number
- EP3714014B1 EP3714014B1 EP18804012.5A EP18804012A EP3714014B1 EP 3714014 B1 EP3714014 B1 EP 3714014B1 EP 18804012 A EP18804012 A EP 18804012A EP 3714014 B1 EP3714014 B1 EP 3714014B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- gelatin
- less
- hydrolysate
- temperature
- endotoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920000159 gelatin Polymers 0.000 title claims description 87
- 235000019322 gelatine Nutrition 0.000 title claims description 87
- 235000011852 gelatine desserts Nutrition 0.000 title claims description 83
- 108010010803 Gelatin Proteins 0.000 title claims description 82
- 239000008273 gelatin Substances 0.000 title claims description 81
- 239000002158 endotoxin Substances 0.000 title claims description 58
- 238000000034 method Methods 0.000 title claims description 36
- 239000000413 hydrolysate Substances 0.000 title claims description 27
- 238000002360 preparation method Methods 0.000 title claims description 10
- 230000003247 decreasing effect Effects 0.000 claims description 4
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 26
- 230000007062 hydrolysis Effects 0.000 description 13
- 238000006460 hydrolysis reaction Methods 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000004094 surface-active agent Substances 0.000 description 8
- 239000000499 gel Substances 0.000 description 7
- 239000001828 Gelatine Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 description 4
- 239000003463 adsorbent Substances 0.000 description 4
- 239000012736 aqueous medium Substances 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000239218 Limulus Species 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000954 titration curve Methods 0.000 description 2
- 241001529572 Chaceon affinis Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 241000239220 Limulus polyphemus Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 108010072542 endotoxin binding proteins Proteins 0.000 description 1
- 238000011013 endotoxin removal Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000416 hydrocolloid Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 238000012454 limulus amebocyte lysate test Methods 0.000 description 1
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000003058 plasma substitute Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000006794 tachycardia Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09H—PREPARATION OF GLUE OR GELATINE
- C09H3/00—Isolation of glue or gelatine from raw materials, e.g. by extracting, by heating
- C09H3/02—Purification of solutions of gelatine
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
- C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
- C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
- C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin
Definitions
- the invention relates to a method for the preparation of gelatin hydrolysate having a low lipopolysaccharide (LPS) content.
- LPS lipopolysaccharide
- Gelatin is a mixture of water-soluble proteins derived from collagen. Gelatin is obtained e.g. by partial hydrolysis of collagen, obtained by aqueous extraction of skin, tendons, ligaments, bones etc. in acid or alkali conditions, or by enzymatic hydrolysis. Gelatin obtained by acid treatment is called Type A gelatin, whereas Type B gelatin is derived from alkali based process.
- Gelatin does not constitute a uniform protein molecule, but comprises a variable amount of protein molecules of variable length, having an average molecular weight of up to 200-250 kDa. Therefore, the molecular weight distribution of gelatin is an important parameter responsible for or determining often critical and important gelatin properties such as viscosity and bloom value, or gel strength.
- Gelatin forms a thermoreversible gel at room temperature, and dissolves in hot water.
- Gelatin is commonly used in diverse industries, for example in food, pharmaceuticals and cosmetics applications, among others as gelling agent and texturizer in e.g. fruit gums and gelatin desserts, but also finds application in de medical field, e.g. for plasma substitution and gelatin based implants.
- the molecular weight varies among others due to different extraction temperatures and conditions. As a result also bloom and viscosity will vary. Temperature is an important parameter in gelatin preparation, e.g. purification conditions before the gelatin can be applied in food, pharmaceutical, technical and medical applications and often needs careful control. When it comes to use of gelatin, in applications where gelling characteristics and viscosity are important, a temperature of 60 °C is considered as maximum handling temperature, although temperatures of up to e.g. 62 °C or 65 °C for a limited time period of e.g. 5 or 10 to 30 or 45 min. may be acceptable under circumstances when some loss of gelling capacity and/or viscosity is tolerated.
- the molecular weight distribution of gelatin is usually measured by size exclusion HPLC (high performance liquid chromatography) techniques, and eluted fractions are detected by UV adsorption and the measured data are evaluated by suitable software, all techniques, known in the art, see e.g. Olijve et.al., Journal of Colloid and Interface Science (2001) 243, 476-482 .
- HPLC high performance liquid chromatography
- eluted fractions are detected by UV adsorption and the measured data are evaluated by suitable software, all techniques, known in the art, see e.g. Olijve et.al., Journal of Colloid and Interface Science (2001) 243, 476-482 .
- a separation column such as TSKgel2000SWXL (Tosoh BioScience, Japan), to obtain high resolution ( Zhang et.al., Food Hydrocolloids 23 (2009) 2001-2007 ).
- Viscosity of gelatin (the dynamic viscosity) is usually measured by measuring the flow time of a 6.67 w/w% solution of gelatin through a standard flow pipet at 60°C, see GME Monograph Standardized Methods for the testing of Edible Gelatin, version 10, 2014 (GME, Brussels, Belgium), herein also referred to as 'GME10', chapter 2.4.2, p. 81 - 86.
- the gel strength of a 6.67 w/w% gelatin gel can be determined by standardized apparatuses (see GME10), such as a QTS 25 Texture Analyzer (Brookfield Viscometers) or a Texture Analyzer TA-XT2 (Stable Micro Systems Ltd., London, United Kingdom), and is indicated by a bloom number (also referred herein as 'bloom value', see GME10).
- hydrolysis of gelatin occurs, i.e. breakdown of protein molecules to smaller peptides, resulting in a lower gel strength or even loss of gelling capacity.
- ⁇ hydrolysed gelatin' is a peptide preparation originating from hydrolysis of gelatin to peptide molecules having an average molecular weight of 70kDa or less, usually 20 kDa or less, usually between 100 and 15000Da. Because of the relatively small molecules, hydrolysed gelatin has no gelling properties, i.e. not capable of gel formation when kept at 0°C for 6 hours (Pharmacopeia definition). Hydrolysed gelatin is e.g.
- collagen used as texture conditioner and moisturizer in topical crèmes, and is also used in nutritional products because of the high glycine, proline and hydroxyproline content and is associated with health effect and it is more and more used for biomedical applications.
- Gelatin hydrolysate is usually prepared by enzymatic hydrolysis of gelatin.
- LPS lipopolysaccharides
- Lipopolysaccharides are found in the outer membrane of Gram-negative bacteria and are potential toxins. LPS are also known as "endotoxins" as lipopolysaccharides are not secreted by bacteria but are part of the membrane structure. Lipopolysaccharides are therefore mainly released after death and lysis of the bacterial cell.
- LPS consist of a variable polysaccharide chain and a lipid moiety, lipid A.
- LPS molecules are about 10 kDa in size, but can form large aggregates in aqueous media, also named "micelles" having a molecular weight of up to 1000 kDa.
- LPS are toxic to most mammals and the animal host will often suffer from a wide spectrum of non-specific pathophysiological reactions, such as fever, tachycardia, organ dysfunction and even death.
- the endotoxin level should preferably be lower than 20, more preferably 10 EU/g, even more preferably less than 6, 5, or even less than 4 EU/g.
- USA governmental regulations of the Food and Drug Administration (FDA) allow a maximum of 0.5 EU/ml or 20EU/device for products that are in contact with the cardiovascular and/or lymphatic system.
- FDA Food and Drug Administration
- the limit is even 0.06 EU/ml or 2.15 EU/device ( ⁇ 2EU/g gelatin).
- an even lower endotoxin limit may apply.
- the Limulus assay is a well-known bioassay in the art to measure up to sub-picogram quantities of LPS.
- Limulus amoebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes ) from the horseshoe crab , Limulus polyphemus.
- LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria . This reaction is the basis of the LAL test, which is then used for the detection and quantification of bacterial endotoxins.
- WO 2009/154440 describes a method for the removal of LPS from a gelatin source wherein the initial endotoxin level of 13,200 EU/g is decreased 10 2.1 fold, i.e. to a value of 105 EU/g. Although this decrease is significant, the levels are still unacceptably high for medical purposes.
- a 30 w/w% aqueous gelatin solution was heated to 90°C in the presence of the surfactant Triton X-100, i.e.
- the solution is brought to conditions such, that the temperature of the solution comprising the gelatin, surfactant, adsorbent and the LPS is above the cloud point temperature of the surfactant at that conditions, in order to allow the surfactant to aggregate so that the aggregates can be removed, together with the LPS adsorbed to the adsorbent, by centrifugation.
- WO 2016/085345A1 describes a method of removing lipopolysaccharide from an aqueous medium comprising gelatin and lipopolysaccharides, said method comprising the steps of providing an aqueous medium comprising gelatin and lipopolysaccharides, adding to the aqueous medium a micelle-forming surfactant.
- EP1829946A1 relates to a method of manufacturing gelatin with reduced endotoxin content.
- the method comprises processing a starting material gelatin-containing solution containing gelatin and an endotoxin with an ultrafiltration film having a molecular weight cut-off falling within a range of from 20,000 to 300,000.
- the present disclosure provides a method for the preparation of a gelatin hydrolysate having a decreased endotoxin content, comprising the steps of:
- the term 'decreased endotoxin content' intends to mean that the endotoxin level of the gelatin hydrolysate of step b., is significantly lower than that of the starting material, i.e. the gelatin or the gelatin hydrolysate before being subjected to step a.
- the level is preferably at least 10 times lower, more preferably at least 100 times lower, more preferably at least 500 times lower or even 700 times lower, more preferably 1000 times or even 5,000 times, or even 10,000 times lower. As will be seen in the examples, a level of 15,000 times lower can be achieved.
- the temperature is between 70 and 125°C, wherein it is to be understood that at temperatures above 100°C, the heating takes place under pressure. Therefore, the temperature is preferably between 80°C and 100°C, more preferably between 90 and 100°C.
- the pH is 3.5 or lower, and the time period is preferably at least 15 minutes.
- the incubation period is chosen such, that the envisaged endotoxin content is reached, while providing a gelatine hydrolysate of the envisaged properties like molecular weight distribution and average molecular weight.
- the skilled person will readily adjust he parameters of temperature, pH and time to arrive at the envisaged hydrolysate having the required low endotoxin content.
- the temperature is preferably between 92 - 98°C, more preferably between 94 - 96°C, while the pH is preferably 3.0 or less, more preferably 2.7 or less, more preferably 2.5 or less, even more preferably between 1.8 - 2.2, and most preferably about 2.0.
- the term 'about' intends to mean that the pH may vary around the value of 2.0, by 0.5 or less, i.e.
- the pH may be chosen in the upper limits as described, e.g. having a value of 3 - 3.5. Further lowering of the pH can be desired to decrease the LPS content even further.
- the time period is preferably 30 minutes or more. In that period, the gelatin hydrolyses at such high temperatures and the endotoxin levels will be attractively low.
- the time period is 1 - 5 hours, preferably 1.5 - 3 hours, more preferably 2 - 2.5 hours.
- the method is free of an enzymatic treatment step.
- enzymes which need to be inactivated and might also require subsequent purification of the hydrolysate, as any foreign protein material such as such enzymes may cause an immune response.
- the product is preferably enzyme-free.
- the method can start with a gelatin hydrolysate, it is preferred to incubate a gelatin solution in step a, that has a molecular weight of more than 70 kDa. As the method conditions have hydrolyzing power for the gelatin, it is not necessary to hydrolyze the gelatin in a preceding step.
- the gelatin hydrolysate in step b. preferably has a molecular weight of 30 kDa or less, preferably of 20 kDa or less, more preferably of 10 kDa or les, even more preferably of 5 kDa or less, or even 4 kDa or less. As discussed above, the gelatine hydrolysate has no gelling power.
- the gelatin hydrolysate in step b. preferably has an endotoxin level of 20 EU/g gelatin hydrolysate or less, preferably of 10 EU/g or less, more preferably of 5 EU/g or less, even more preferably of 2 EU/g or less, most preferably of 1EU/g or less.
- Such values can be obtained with gelatin starting materials having 10,000 - 15,000 EU/g gelatin or more.
- gelatin hydrolysate obtainable by the method of any of the preceding claims.
- Such gelatin hydrolysate preferably has a molecular weight of 20 kDa or less, preferably of 15 kDa or less, and an endotoxin level of 20 EU/g gelatin hydrolysate or less, preferably of 10 EU/g or less, more preferably of 5 EU/g or less, even more preferably of 2 EU/g or less, most preferably of 1EU/g or less, in particular measured according to the above-described LAL method.
- the molecular weight has been measured according to the method described by Zhang, supra, and is shown in tables 2 and 3.
- Table 2 molecular weight at 95°C at different pH pH 2.0 and 95°C pH 2.5 and 95°C pH 2.7 and 95°C Time (h) MW (Da) MW (Da) MW (Da) 0 155 155 155 1 9.5 11.8 12.0 2 7.0 8.8 8.5 3 6.4 7.4 7.5 4 5.4 6.7 6.7
- Table 3 molecular weight at pH 2.0 at different temperature pH 2.0 and 80°C pH 2.0 and 90°C pH 2.0 and 95°C Time (h) MW (Da) MW (Da) MW (Da) 0 150 150 155 13.8 9.5 9.5 2 12.8 7.4 7.0 3 11.2 6.3 6.4 4 10.6 5.6 5.4
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Claims (11)
- Verfahren zur Herstellung eines Gelatinehydrolysats mit einem verminderten Endotoxingehalt, umfassend die Schritte:a. Inkubieren einer Lösung von Gelatine von Gelatinehydrolysat bei einer Temperatur von 70 - 125°C und einem pH-Wert von 3,5 oder kleiner über einen Zeitraum von wenigstens 15 Minuten undb. Gewinnen des Gelatinehydrolysats.
- Verfahren nach Anspruch 1, wobei die Temperatur zwischen 90 - 100°C liegt.
- Verfahren nach Anspruch 1, wobei die Temperatur zwischen 92 - 98°C, bevorzugt zwischen 94 - 96°C liegt.
- Verfahren nach einem der vorhergehenden Ansprüche, wobei der pH-Wert bei 2,7 oder darunter liegt.
- Verfahren nach Anspruch 4, wobei der pH-Wert bei 3,0 oder darunter, bevorzugt bei 2,5 oder darunter, bevorzugt zwischen 1,8 - 2,2, am meisten bevorzugt bei etwa 2,0 liegt.
- Verfahren nach einem der vorhergehenden Ansprüche, wobei der Zeitraum 30 Minuten lang oder länger ist.
- Verfahren nach Anspruch 6, wobei der Zeitraum 1 - 5 Stunden, bevorzugt 1,5 - 3 Stunden, bevorzugter 2 - 2,5 Stunden lang ist.
- Verfahren nach einem der vorhergehenden Ansprüche, das frei von einem enzymatischen Behandlungsschritt ist.
- Verfahren nach einem der vorhergehenden Ansprüche, wobei in Schritt a. eine Gelatinelösung inkubiert wird, wobei die Gelatine ein Molekulargewicht größer 70 kDa aufweist.
- Verfahren nach einem der vorhergehenden Ansprüche, wobei das Gelatinehydrolysat in Schritt b. ein Molekulargewicht von 30 kDa oder kleiner, bevorzugt von 20 kDa oder kleiner, bevorzugter von 10 kDa oder kleiner, noch bevorzugter von 5 kDa oder kleiner aufweist.
- Verfahren nach einem der vorhergehenden Ansprüche, wobei das Gelatinehydrolysat in Schritt b. ein Endotoxinniveau von 20 EU/g Gelatinehydrolysat oder kleiner, bevorzugt von 10 EU/g oder kleiner, bevorzugter von 5 EU/g oder kleiner, noch bevorzugter von 2 EU/g oder kleiner, am meisten bevorzugt von 1 EU/g oder kleiner aufweist.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP17203341.7A EP3489314A1 (de) | 2017-11-23 | 2017-11-23 | Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt |
PCT/EP2018/082238 WO2019101864A1 (en) | 2017-11-23 | 2018-11-22 | Method for the preparation of gelatin hydrolysate having a low endotoxin content |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3714014A1 EP3714014A1 (de) | 2020-09-30 |
EP3714014B1 true EP3714014B1 (de) | 2024-01-10 |
Family
ID=60661711
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP17203341.7A Withdrawn EP3489314A1 (de) | 2017-11-23 | 2017-11-23 | Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt |
EP18804012.5A Active EP3714014B1 (de) | 2017-11-23 | 2018-11-22 | Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP17203341.7A Withdrawn EP3489314A1 (de) | 2017-11-23 | 2017-11-23 | Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt |
Country Status (12)
Country | Link |
---|---|
US (2) | US11725118B2 (de) |
EP (2) | EP3489314A1 (de) |
JP (1) | JP2021504463A (de) |
KR (1) | KR20200096250A (de) |
CN (1) | CN111630124B (de) |
AU (1) | AU2018373882B2 (de) |
BR (1) | BR112020010267A2 (de) |
CA (1) | CA3082946A1 (de) |
ES (1) | ES2969654T3 (de) |
IL (1) | IL274818A (de) |
MX (1) | MX2020005280A (de) |
WO (1) | WO2019101864A1 (de) |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06206893A (ja) * | 1992-09-07 | 1994-07-26 | Suntory Ltd | 新規なジサッカライド誘導体 |
US6992172B1 (en) * | 1999-11-12 | 2006-01-31 | Fibrogen, Inc. | Recombinant gelatins |
EP1232262B1 (de) * | 1999-11-12 | 2008-08-20 | Fibrogen, Inc. | Rekombinantes gelatin in impstoffen |
US7485323B2 (en) * | 2005-05-31 | 2009-02-03 | Gelita Ag | Process for making a low molecular weight gelatine hydrolysate and gelatine hydrolysate compositions |
JP4404866B2 (ja) * | 2006-03-03 | 2010-01-27 | ゼライス株式会社 | エンドトキシン含有量を低減したゼラチンの製造方法および低エンドトキシンゼラチン |
ES2621452T3 (es) | 2008-06-19 | 2017-07-04 | Bender Analytical Holding B.V. | Método para retirar impurezas de material biopolimérico |
US20140193463A1 (en) * | 2013-01-04 | 2014-07-10 | China Medical University | Peptide for inhibiting dipeptidyl-peptidase iv |
NL2013880B1 (en) * | 2014-11-26 | 2016-10-11 | Rousselot B V | Gelatin purification. |
-
2017
- 2017-11-23 EP EP17203341.7A patent/EP3489314A1/de not_active Withdrawn
-
2018
- 2018-11-22 MX MX2020005280A patent/MX2020005280A/es unknown
- 2018-11-22 US US16/766,654 patent/US11725118B2/en active Active
- 2018-11-22 BR BR112020010267-9A patent/BR112020010267A2/pt unknown
- 2018-11-22 EP EP18804012.5A patent/EP3714014B1/de active Active
- 2018-11-22 AU AU2018373882A patent/AU2018373882B2/en active Active
- 2018-11-22 WO PCT/EP2018/082238 patent/WO2019101864A1/en unknown
- 2018-11-22 CA CA3082946A patent/CA3082946A1/en active Pending
- 2018-11-22 JP JP2020545871A patent/JP2021504463A/ja active Pending
- 2018-11-22 CN CN201880087138.6A patent/CN111630124B/zh active Active
- 2018-11-22 ES ES18804012T patent/ES2969654T3/es active Active
- 2018-11-22 KR KR1020207018184A patent/KR20200096250A/ko unknown
-
2020
- 2020-05-20 IL IL274818A patent/IL274818A/en unknown
-
2023
- 2023-08-08 US US18/231,468 patent/US20240034909A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
IL274818A (en) | 2020-07-30 |
US20240034909A1 (en) | 2024-02-01 |
WO2019101864A1 (en) | 2019-05-31 |
JP2021504463A (ja) | 2021-02-15 |
CA3082946A1 (en) | 2019-05-31 |
KR20200096250A (ko) | 2020-08-11 |
US20200362201A1 (en) | 2020-11-19 |
CN111630124B (zh) | 2023-05-16 |
AU2018373882B2 (en) | 2023-11-09 |
AU2018373882A1 (en) | 2020-06-11 |
ES2969654T3 (es) | 2024-05-21 |
EP3714014A1 (de) | 2020-09-30 |
MX2020005280A (es) | 2020-10-07 |
BR112020010267A2 (pt) | 2020-11-17 |
CN111630124A (zh) | 2020-09-04 |
EP3489314A1 (de) | 2019-05-29 |
US11725118B2 (en) | 2023-08-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109790561B (zh) | 利用基本上不含凝固蛋白原的鲎阿米巴样细胞溶解物检测内毒素的方法 | |
JP7009586B2 (ja) | ゼラチン精製 | |
Shi et al. | Effect of caseinate glycation with oligochitosan and transglutaminase on the intestinal barrier function of the tryptic caseinate digest in IEC-6 cells | |
Mouecoucou et al. | In vitro allergenicity of peanut after hydrolysis in the presence of polysaccharides | |
EP3714014B1 (de) | Verfahren zur herstellung von gelatinehydrolysat mit niedrigem endotoxingehalt | |
Kim et al. | Structural features of glycoprotein purified from Saccharina japonica and its effects on the selected probiotic properties of Lactobacillus plantarum in Caco-2 cell | |
Xia et al. | Immune activity of sweet potato (Ipomoea batatas L.) glycoprotein after enzymatic and chemical modifications | |
Vojdani | Immune reactivities against gums | |
Zimoch-Korzycka et al. | Chitosan and cystatin/lysozyme preparation as protective edible films components | |
Setiasih et al. | Dissolution study of purified bromelain from pineapple cores (Ananas comosus [L.] Merr) encapsulated in alginate-chitosan microcapsule | |
JP7057939B2 (ja) | ローヤルゼリー素材の製造方法、ローヤルゼリー素材、ローヤルゼリー含有飲食品、及びローヤルゼリー含有化粧料 | |
Il'ina et al. | Determination of residual protein and endotoxins in chitosan | |
US2432970A (en) | Method of reducing the pyrogen contents of parenteral solutions | |
CN116253809A (zh) | 一种去除壳聚糖中内毒素的方法 | |
Xinyu et al. | A review on glycosylation of protein catalyzed by transglutaminase with chitosan/chitosan derivatives | |
Du et al. | Focusing on papain release in the intestine: The effects of Chitinous materials on alginate microsphere properties | |
Zimoch-Korzycka et al. | Research Article Chitosan and Cystatin/Lysozyme Preparation as Protective Edible Films Components | |
JPS63230038A (ja) | 塩存在下において加熱透明液、及び透明ゲルを形成する調整卵白及びその製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20190215 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: BA ME |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
P01 | Opt-out of the competence of the unified patent court (upc) registered |
Effective date: 20230525 |
|
17Q | First examination report despatched |
Effective date: 20230619 |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
INTG | Intention to grant announced |
Effective date: 20231013 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE PATENT HAS BEEN GRANTED |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602018064034 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: FP |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG9D |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: FG2A Ref document number: 2969654 Country of ref document: ES Kind code of ref document: T3 Effective date: 20240521 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 1648885 Country of ref document: AT Kind code of ref document: T Effective date: 20240110 |